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Biomedical subjects

T Greve

Publications and source records attributed to T Greve.

At least 73 records · Page 4Linked to original sources
PubMed1997

Comparison of two manipulation methods to produce in vitro fertilized, biopsied and vitrified bovine embryos.

The objective of this study was to compare the overall efficiency, measured by in vitro embryonic survival, and practical value of bovine in vitro embryo production, biopsy, vitrification, and direct transfer technology using 2 different manipulation methods for biopsy. Slaughterhouse-derived oocytes were matured in vitro, fertilized (Day 0) with frozen-thawed, Percoll-separated spermatozoa and cultured on a granulosa cell monolayer. In Experiment 1, one or two blastomeres were expelled from Day 4 embryos by mechanical force through a hole made by partial zona dissection. Using a darning needle hole system for individual culture of biopsied embryos from Day 4 to Day 7.5, the blastocyst per oocyte rate was 50%, and 76% of the blastocysts survived subsequent vitrification and direct in-straw rehydration. Attempts to increase the cell number of the biopsies by further in vitro culture were unsuccessful. In Experiment 2, Day 7 and Day 8 embryos were manually biopsied before or after vitrification. When biopsy was performed before vitrification, 98% of the embryos survived manipulation, and 86% of these re-expanded after vitrification and in-straw dilution. Biopsy after vitrification was less efficient, since only 69% of the embryos survived both processes. The cumulative efficiency of embryo production, Day 7.5 biopsy and vitrification--in-straw direct rehydration was lower (P < 0.001) than that of Day 4 biopsy and Day 7.5 vitrification (29 vs 38%, respectively). However, a Day 7.5 biopsy may have the more practical application since the size of the biopsy is larger and the process is not as time-consuming as the long-term individual culture of the biopsied embryos.

Journal Article↗
PubMed1997

Nucleus ultrastructure and transcriptional activity of bovine oocytes in preantral and early antral follicles.

An understanding of the recruitment and growth of follicles within the bovine ovary is crucial to their successful exploitation in vitro. The aim of the present study was to describe the nuclear ultrastructure and transcriptional activity of primordial to early tertiary follicular oocytes from bovine adult ovaries. Small blocks of ovarian cortex were incubated in medium enriched with 3H-uridine for 30 min. Subsequently, the tissue blocks were fixed in Karnowsky's fixative, dehydrated, epon embedded, sectioned (2 microns), processed for autoradiography, and examined under light microscopy. Sections showing preantral follicles with presumptive oocyte nucleoli were reembedded for transmission electron microscopy. The follicles were divided into five categories: 1) resting primordial, with a single layer of flattened granulosa cells, 2) activated primordial, with a single layer of flattened and some cuboidal granulosa cells, 3) primary, with a single layer of cuboidal granulosa cells, 4) secondary, with a complete or incomplete bilayer of cuboidal cells, and 5) tertiary, with more than two layers of granulosa cells delineating one or more intercellular cavities. The granulosa cells of all follicle classes were transcriptionally active. However, the oocytes did not display transcriptional activity, as measured by the present means, until the secondary and tertiary follicular stages. The oocyte nucleolus was granular in the primordial follicles. Following follicular activation, fibrillar centres invaded the nucleolus and, in the early tertiary follicle, numerous fibrillar centres were distributed throughout the nucleolus. In conclusion, the oocyte nucleolar function is gradually activated at follicle activation, and oocyte transcription is initiated at approximately the time of the secondary follicle stage.

Animals↗
PubMed1997

Oocyte ultrastructure in bovine primordial to early tertiary follicles.

The aim of the present study was to describe in detail the changes occurring in the cytoplasmic ultrastructure of the bovine oocyte from the onset of growth in the primordial follicle until the completion of growth in the tertiary follicle. Bovine oocytes from primordial, primary, secondary and early to mid-antral follicles were processed and analysed by light and transmission electron microscopy. The primordial follicular oocyte was characterized by numerous coated pits on the oolemma and the accumulation of free and organelle-related smooth (SER) and rough (RER) endoplasmic reticulum, round mitochondria and Golgi complexes around the nucleus, which was located slightly off centre. Up to the secondary follicular stage the oocyte displayed an increase in the number of microvilli, elongated mitochondria and Golgi complexes. During the secondary follicular stage, formation of the zona pellucida, development of gap junctions between the oocyte and the granulosa cells, formation of the cortical granules in the oocyte and reduction in the number of coated pits on the oolemma were seen. In the tertiary follicular oocyte up to 100 microm in diameter, the number of Golgi complexes and lipid droplets increased and the organelles were dislocated to the deep cortical region. During the final growth of the oocyte up to >120 microm, the organelles were dislocated further to the peripheral region, the extent of the free SER and RER compartments were reduced, the number of individual cortical granules increased, hooded mitochondria became abundant and the perivitelline space developed. In conclusion, the growth of the bovine oocyte is associated with the relocation and modulation of a number of cytoplasmic organelles as well as the development of oocyte specific structures such as the zona pellucida and cortical granules.

Animals↗
PubMed1997

Superovulatory response of dairy cattle (Bos taurus ) in a tropical environment.

Dairy (Bos taurus) heifers and cows (n = 40) in a tropical environment were treated during mid-luteal phase using either SUPER-OV(R) or OVAGEN to induce superovulatory response after synchronization of the superovulatory estrus with a synthetic progestagen and cloprostenol (PG). Estrous cattle were inseminated twice using frozen-thawed semen, and embryos were recovered nonsurgically, on-farm, 7 d later. Between initiation of gonadotrophin treatment and recovery of embryos, 4 blood samples per animal were collected from 26 animals for determination of plasma progesterone (P4) concentration. Two (5%), 28 (70%) and 10 (22%) of the animals were observed in estrus 1.5, 2 and 2.5 to 3 d after PG, respectively. There was no difference (P = 0.7) in the number of palpable CL between animals treated with SUPER-OV (7.6 +/- 1.0; n = 18) and those treated with OVAGEN (7.9 +/- 1.1; n = 22). There was also no significant difference (P > 0.05) between Jersey vs Ayrshire breeds or heifers vs cows in the ovarian response as estimated by the number of palpable CL. However, a higher proportion of Ayrshire cattle and donors treated with OVAGEN yielded a higher total number and viable/transferable embryos than Jersey and SUPER-OV-treated cattle. There was a significant (P < 0.05) correlation between the number of CL and total number of embryos (r = 0.65); the number of transferable embryos was also significantly related to the total number of embryos per recovery (r = 0.85; P < 0.05). For 15 animals with normal P4 profiles, the mean (+/-SEM) plasma P4 concentration was 14.4 +/- 0.8, 0.5 +/- 0.2, 5.4 +/- 1.1 and 39.4 +/- 3.0 nmol L at initiation of gonadotrophin treatment, superovulatory estrus and Days 3 and 7, respectively. The mean (+/-SEM) interval between a PG injection given after embryo recovery and the induced estrus was 7.1 +/- 0.7 d (range 3 to 14 d) and the length of the superovulatory cycle was 24.1 +/- 3.2 d (range 12 to 35 d).

Journal Article↗
PubMed1997

Are lower fertility bulls necessarily less fertile? Proposals concerning insemination procedures.

This essay argues that current procedures of selection for high fertility bulls may overlook young males of potentially high fertility unless these are tested by modified procedures of insemination. Should this suggestion prove to be true, even if only for a small proportion of young bulls that would not previously have been retained as stud animals, then valuable production genes would be kept in the national herd. Modified procedures of introducing the sperm suspension might include (I) deep intra-uterine insemination, (II) insemination into the functional sperm reservoir in the Fallopian tube isthmus, (III) laparoscopic insemination close to the utero-tubal junction, (IV) intra-peritoneal insemination, (V) insemination under conditions of mild superovulation, and (VI) insemination with smooth muscle stimulants and/or sperm stimulating agents added to the suspension. (VII) The potential value of in vitro fertilization assays, such as the zona-free hamster oocyte sperm incorporation test, is also noted. Even if only one of these approaches were found to be fruitful, its impact could be of major significance for the cattle breeding industry.

Animals↗
PubMed1997

Graafian follicles are cooler than neighbouring ovarian tissues and deep rectal temperatures.

In order to establish appropriate culture temperatures for in-vitro maturation of pig ovarian oocytes, large Graafian follicles (7-10 mm diameter) were sensed by infra-red technology during the latter part of a spontaneous oestrous cycle. Temperatures were measured under systemic anaesthesia almost instantaneously upon revealing the ovaries at mid-ventral laparotomy. Temperature differentials were observed within all 16 ovaries sensed in 14 animals. Ovaries were always cooler than deep rectal temperatures (mean rectal temperature was 38.0 +/- 0.4 degrees C; range 37.5-38.6 degrees C) and mature follicles always cooler than ovarian stroma (35.6 +/- 0.3 degrees C versus 37.3 +/- 0.2 degrees C respectively; P < 0.01). Such follicles were frequently 1.5-1.8 degrees C cooler than the adjacent stroma, the mean being 1.7 +/- 0.4 degrees C. Small Graafian follicles (< 5-6 mm diameter) and recent ovulations did not show this differential. The control experiment of excising an ovary, deep freezing it in liquid nitrogen, and then restoring it to the body cavity before further sensing indicated that intra-ovarian temperature gradients depended on the activity of living tissues and/or a functional blood supply. Furthermore, calculation of anticipated rates of cooling for exposed Graafian follicles strongly suggested that artefacts could not have been solely responsible for the observed temperatures. Endothermic reactions within mature follicles were thus brought into focus. It is concluded that follicular temperatures may influence the meiotic progression and cytoplasmic maturation of oocytes and act to regulate enzymatic activity in the biosynthetic pathways for steroid and/or peptide hormones.

Animals↗
PubMed1997

Time course of pronuclear deoxyribonucleic acid synthesis in parthenogenetically activated bovine oocytes.

The progress of pronuclear DNA synthesis was monitored by the radioactive precursor 3H-thymidine during the first cell cycle of parthenogenetically activated bovine oocytes. Bovine oocytes were exposed to Ca2+ ionophore A23187 at 24, 30, or 36 h after the onset of in vitro maturation. Young 24-h oocytes were subsequently cultured for 6 h in the protein synthesis inhibitor, cycloheximide (CHX), to ensure similar rates of activation (96-100%) and pronuclear formation (93-97%) among all groups of oocytes. Subsequent autoradiographic experiments revealed a slightly, but not significantly, accelerated start of DNA synthesis in aged (36 h) oocytes. Maximum levels of DNA labeling were reached within 4 h regardless of oocyte maturation age and persisted for 4 h in 30-h oocytes compared to 2 h in 36-h and 24-h oocytes. The period of DNA synthesis lasted for a total of 12-14 h in all groups of oocytes, and the duration of S-phase was less than 6 h. Since rates of pronuclear formation (58%) and labeling (58%) corresponded to each other, it is argued that only a fully developed pronucleus can synthesize DNA. Oocyte labeling performed in the presence of CHX revealed the capability of CHX to inhibit DNA synthesis up to 8 h postactivation. Removal of CHX by washing when the majority (94%) of oocytes had formed a fully developed pronucleus (at 8 h postactivation) led to the synchronous start of DNA synthesis within 1.5-2 h post-CHX culture. This concomitantly defined the time required for synthesis of vital proteins needed for the entry into S-phase and/or DNA replication. The prolonged exposure of activated oocytes to CHX (10-12 h) negatively affected the pattern of DNA synthesis. The start of DNA synthesis was postponed and reduced pronuclear labeling was observed. In addition, CHX-treated oocytes often exhibited a characteristic punctate pattern of pronuclear labeling in which silver grains were accumulated into clusters. In conclusion, the present results provide knowledge about timing and a possible synchronization of DNA synthesis in parthenogenetically activated bovine oocytes.

Animals↗
PubMed1997

Intracytoplasmic sperm injection of in vitro-matured equine oocytes.

Intracytoplasmic sperm injection (ICSI) was performed on equine oocytes matured in vitro. The oocytes were aspirated from abattoir ovaries and matured in vitro for 36 h at 38 degrees C. ICSI was performed using frozen/thawed stallion semen after swimup in medium containing human serum albumin. Sperm-injected oocytes were either 1) cultured in vitro for 10, 20, or 72 h; 2) transferred to oviducts of pseudopregnant mice; or 3) transferred to a synchronized mare after initial in vitro culture. The transferred ova were recovered after 72 h, and all ova were subsequently fixed, stained, and processed for light and transmission electron microscopy. Single pronucleus formation was observed in 2 out of 12 presumptive zygotes 10 h postinjection, at which time abundant cortical granules were observed in the subplasmalemmal region. Twenty hours postinjection, however, 2 pronuclei were observed in 6 of 12 injected oocytes (fertilization rate 50%), and almost all cortical granules were released. The cleavage rate in vitro was 16% after 72 h in culture, and the most advanced embryo stages obtained were 6- to 8-cell embryos. The cleavage rate in vivo was very low since only 1 of 10 recovered had cleaved to the 2-cell stage. Thus, in conclusion, ICSI fertilization of equine oocytes did result in fertilization, pronucleus formation, and cortical granule release. However, the observed fertilization rate and oocyte activation was not paralleled by substantial cleavage of the zygotes.

Animals↗
PubMed1997

Survival and development of bovine blastocysts produced in vitro after assisted hatching, vitrification and in-straw direct rehydration.

The purpose of this study was to establish an efficient combination of assisted hatching and cryopreservation procedures for producing bovine embryos in vitro. A total of 1312 day 7 blastocysts were subjected randomly to 14 different combinations of three factors: osmotic stress, assisted hatching and vitrification. Re-expansion, initiation and completion of the hatching process, as well as attachment to the culture dish, were analysed by SAS Genmod procedure. Incubation with sucrose was found to decrease survival rates; among the assisted hatching procedures used, zona fenestration resulted in higher survival rates compared with partial zona dissection and controls; and vitrification decreased survival and further development. The combined effect of sucrose incubation and vitrification decreased further development markedly, as did partial zona dissection followed by vitrification. Partial zona dissection performed in medium containing sucrose severely lowered embryo survival. Zona fenestration without sucrose incubation followed by vitrification did not compromise further embryo development: 86%, 84% and 79% of the blastocysts initiated, completed hatching and attached to the bottom, respectively. These data were not different from the controls (80%, 76% and 63%, respectively; P > 0.05). Cell count analysis revealed a decrease in the total number of cells as a result of the assisted hatching and vitrification compared with controls (135 versus 202, respectively; P < 0.0001). Although embryo transfer results (36% pregnancy rate and 30% calving rate) require further improvement, this combination of methods may prove useful in the commercial production of bovine embryos in vitro.

Animals↗
PubMed1997

Establishment of the block against sperm penetration in parthenogenetically activated bovine oocytes matured in vitro.

The ability of a single electric pulse to mimic a block against sperm penetration in bovine oocytes matured in vitro was investigated. Confocal laser scanning microscopy detected a global loss of spots, presumed to be cortical granules, stained with Lens culinaris agglutinin, in pulsed oocytes. Transmission electron microscopy revealed that cortical granule exocytosis occurred within 1 min of stimulation and the number of remaining cortical granules was significantly reduced in all pulsed oocytes. The ability of pulsed oocytes to undergo fertilization in vitro was also affected, as only 31% of the pulsed oocytes were penetrated compared with 87% in the control group. Since incidences of penetration in pulsed oocytes (31%), and of polyspermy in control oocytes (18%) did not differ and were highly correlated (P = 0.009) among trials (n = 15), the induced block is considered to be comparable with the natural block triggered by a spermatozoon. The increased resistance of the zona pellucida to pronase E observed in pulsed oocytes suggests that the induced block depends, at least partly, on modifications of zona pellucida glycoproteins. Finally, the majority (66%) of pulsed, penetrated oocytes did not form male pronuclei, probably as a consequence of asynchrony between the formation of female pronucleus and sperm penetration. The reduced ability of the cytoplasm to induce the formation of a male pronucleus was accompanied by a fall in histone H1 kinase activity to basal values by 3 h after stimulation. These results demonstrate that a single electric pulse can induce a block against sperm penetration similar to that of the spermatozoon itself.

Acrosome↗
PubMed1996

Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration.

The aim of this work was to investigate the possibilities of simplification, and to outline the limits of application, of a vitrification method for cow embryos. Morulae and blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed bull semen, and subsequent culture on a granulosa cell monolayer. Vitrification was performed by equilibration of embryos with 12.5% ethylene glycol and 12.5% dimethylsulphoxide at 20-22 degrees C for 60 s, then with 25% ethylene glycol and 25% dimethylsulphoxide at 4 degrees C for another 60 s. Embryos were then loaded in straws, placed in liquid nitrogen vapour for 2 min, and then plunged. Straws were thawed in a 22 degrees C water-bath, the embryos were directly rehydrated and further incubated in straw, and were then expelled and cultured in vitro for 72 h. In the first experiment, embryos of different age and developmental stage (Day 5 compacted morulae, Day 6 early blastocysts, Days 6 and 7 blastocysts, Day 7 expanded blastocysts and Day 8 hatched blastocysts) as well as Days 7 and 5 blastocysts previously subjected to partial zone dissection were vitrified. After thawing, the re-expansion rates of blastocysts and zona-dissected embryos did not differ (67 and 87%, respectively), and hatching was more frequent for blastocysts frozen in advanced developmental stages (34, 47 and 63% for early blastocysts, blastocysts and expanded blastocysts, respectively). The re-expansion rate of morulae was lower (10%) and no hatching of these embryos was observed. In the second experiment, Day 7 expanded blastocysts were vitrified using PBS, PBS+albumin, TCM199 and TCM199+calf serum as holding media. No differences in re-expansion and hatching rates were seen. However, when incubation with the concentrated cryoprotectant solution was performed at 20-22 degrees C, the embryo survival rate decreased (PBS+albumin) or no embryo survived (TCM199+calf serum) the vitrification procedure. In the third experiment, Day 7 expanded blastocysts were vitrified, thawed, cultured for 1 day, and then re-expanded embryos were again vitrified and thawed. Out of the 87% that survived the first cycle, 73% re-expanded and 47% hatched following the second vitrification and thawing. These observations prove that the vitrification procedure described is relatively harmless, that it can be used for blastocysts of different developmental stages and that an intact zona is not required to obtain high survival rates.

Animals↗
PubMed1996

Transcriptional activity in in vitro produced bovine two- and four-cell embryos.

The objectives of this study on in vitro produced bovine two- and four-cell embryos were (1) to investigate the uptake of 3H-uridine through the plasma membrane, (2) to characterize the pattern of RNA synthesis during the second cell cycle, and (3) to measure the incorporation of 3H-uridine into de novo synthesized RNA. A total of 200 embryos were incubated with 3H-uridine for 15, 30, 60 (two- and four-cell embryos), 120 (four-cell embryos), 180 (two-cell embryos), and 240 min (two- and four-cell embryos), respectively. 3H-uridine uptake reached a maximum by 30 min in two-cell embryos, whereas four-cell embryos reached a maximum at 120 min. A total of 440 two-cell embryos were isolated 27-33 hr postinsemination (hpi), and 90 of these were incubated for 10 hr with 3H-uridine (200 microCi/ml). The remainder were incubated with 3H-uridine for 3 hr starting at 0-3 (n = 54), 3-6 (n = 75), 6-9 (n = 77), or 9-12 (n = 77) hr after cleavage to the two-cell stage. Control two-cell embryos (n = 67) were incubated with 3H-uridine supplemented with 5 mg/ml of unlabelled uridine for 10 hr (inhibition control), or they were incubated with 3H-uridine for 10 hr and RNase treated (100 micrograms/ml) post fixation (RNase control). Subsequently, the embryos were processed for autoradiography. The long-term incubation revealed transcription (autoradiographically labelled nuclei) in a total of 77% of the two- and four-cell embryos. No transcription was observed in any of the 3 hr incubation groups. The RNase control embryos lacked labelling of the nuclei, whereas the inhibition control embryos only showed markedly reduced labelling. Finally, total RNA extraction was performed on a total of 336 two-cell embryos that were incubated with 3H-uridine or 3H-uridine supplemented with unlabelled uridine for 2, 5, or 10 hr. It was possible to detect an increasing amount of labelled RNA after the 2, 5, and 10 hr incubation periods, and it was possible to inhibit this incorporation competitively. Together the data demonstrate a low level of transcription during the second cell cycle without a well-defined transcriptional peak.

Animals↗
PubMed1996

Nucleus structure and transcriptional activity in relation to oocyte diameter in cattle.

Bovine abattoir ovaries were sliced, and recovered oocytes were washed and incubated in medium enriched with 3H-uridine for 30 min. Uridine incorporation was stopped by washing at 4 degrees C in PBS supplemented with cold uridine. The oocytes were grouped according to their inside diameter- < 100, 100- < 110, 110- < 120, and > or = 120 microns-and processed for autoradiography and transmission electron microscopy. Oocytes < 110 microns in diameter typically presented fibrillogranular nucleoli and were actively transcribing; in contrast, most oocytes > 110 microns displayed electron-dense fibrillar nucleoli and lacked transcriptional activity, as measured by the present means. Based on morphological and transcriptional information, a dynamic model of nucleolus inactivation is proposed. The degree of chromatin condensation varied among oocytes. Fibrillogranular nucleoli were most frequently accompanied by lightly condensed chromatin. The dense fibrillar nucleoli were usually encapsulated by heavily condensed chromatin. The oocyte nuclei underwent a peripheral translocation as the oocyte diameter increased from < 100 to 110 microns. In conclusion, RNA synthesis appeared to cease as the oocyte diameter exceeded 110 microns, and concomitantly the nucleoli restructured from fibrillogranular to dense fibrillar.

Animals↗
PubMed1996

Overall efficiency of in vitro embryo production and vitrification in cattle.

In 5 replicates a total of 719 immature oocytes recovered from 94 slaughterhouse-derived bovine ovaries were matured and fertilized in vitro, then cultured for 7 to 9 d on a granulosa cell monolayer in TCM 199 supplemented with calf serum. Of 338 blastocysts (47% of oocytes cultured), 301 were vitrified in Hepes/bicarbonate buffered TCM-199 medium, 20% calf serum and dimethylsulfoxide and ethylene glycol as the cryoprotectants. After thawing in 1 M sucrose and subsequent culture in vitro, 237 (79%) of the blastocysts re-expanded and 177 (59%) hatched. Re-expansion and hatching rates differed between the blastocysts vitrified on Day 7 and Day 8 (84 and 69% vs 70 and 41%, respectively). We conclude that the applied methods are relatively simple and inexpensive to use, with an overall efficiency of the in vitro production/vitrification procedure being 1.9 hatched blastocyst/ovary. Therefore, this system seems suitable for large-scale production of cryopreserved bovine embryos for various purposes.

Journal Article↗
PubMed1996

Pregnancies, calves and calf viability after transfer of in vitro produced bovine embryos.

Pregnancy, parturition and calf survival following the transfer of embryos produced in vitro were monitored. A total of 44 blastocysts was transferred in pairs to 1 uterine horn ipsilateral to the corpus luteum (CL) of 22 synchronized heifers. At Day 42 of development 14 recipients (64%) were pregnant; the calving rate was also 64%. The twinning rate was 9/14 at Day 42 and 7/14 at birth, for an overall fetal mortality rate of 9%. The average gestation length was 281 and 275 d for single and twin pregnancies, respectively. Blood samples from recipients were collected for determination of bovine pregnancy associated glycoprotein (bPAG) from 2 wk after transfer and throughout the pregnancy. During the first trimester of pregnancy, the bPAG concentration was significantly higher in twin than in single bearing heifers, and the perinatal increase in bPAG was correlated positively with the total weight of the fetus(es). The percentage of male calves was 43%. The birth weight of twin individuals was 25 +/- 1 kg, which was 78% of the birthweight of the singletons (32 +/- 2 kg). One singleton calf was oversized, weighing 58 kg (80% more than the median weight of the other singletons). Stillbirths occurred in 21% of the twins, butin none of the singletons. Calf mortality during the first 14 d was higher for twins (4/11) than for singletons (1/7) due to infections and cerebellar hypoplasia. Karyotyping the calves detected no cytogenetically recognizable abnormalities. All calves were negative for BVD virus and IBR antibodies. The results of this study showed that although the incidence of fetal loss was low, there was an unacceptable high perinatal mortality of the calves. Thus it is likely that the blood supply through the placenta of animals pregnant with twins was impaired or it is possible that these fetuses and calves had increased stress susceptibility caused by the in vitro conditions. Furthermore, the birth of 1 oversized calf, 2 calves with cerebellar hypoplasia and 5 calves succumbing to infections seems to indicate that a proportion of in vitro produced calves may suffer from factors inherent in the in vitro production system.

Journal Article↗
PubMed1996

Transcription and cell cycle-dependent development of intranuclear bodies and granules in two-cell bovine embryos.

Bovine two-cell embryos were produced by maturation and fertilization in vitro and isolated at 27, 30 or 33 h after insemination. Embryos were incubated with [3H]uridine for 10 h. Other embryos were incubated with [3H]uridine for 1 h starting at 0-3 h, 3-6 h, 6-9 h or 9-12 h after cleavage (hpc) to the two-cell stage. Subsequently, all embryos were washed, fixed, dehydrated, embedded in Epon and sectioned for light microscope autoradiography and transmission electron microscopy. Thus, at the time of fixation the embryos incubated in [3H]uridine for 1 h represented the periods 1-4, 4-7, 7-10 and 10-13 hpc. Among embryos subjected to [3H]-uridine incubation for 10 h, a majority of those in interphase displayed auto-radiographic labelling over the nuclei, whereas none of the embryos incubated for 1 h did so. At 1-4 hpc, two-cell embryos presented electron-dense nucleolus precursor bodies and large clusters of electron-dense granules of various sizes in their nuclei. At 4-7 hpc, ring-shaped or horseshoe-shaped bodies of the same electron density as the nucleolus precursor bodies were found in the periphery of the granule clusters. At 7-10 hpc, several incomplete ring-like bodies of the same electron density as the nucleus precursor bodies were found deeply embedded in the granule clusters. The interior of these bodies contained granules lining a central vacuole. At 10-13 hpc, all two-cell embryos were in mitosis. It is concluded that bovine embryos produced in vitro display a certain rate of transcription during the second cell cycle without the presence of a well-defined transcriptional peak, and that this activity is paralleled by cell cycle-dependent interaction of intranuclear bodies and granules.

Animals↗
PubMed1996

Intersexuality in pigs: clinical, physiological and practical considerations.

Veterinary surgeons and practical pig farmers need to be aware of a condition that can have important deleterious consequences in a breeding herd. The animals in question have sometimes been referred to as hermaphrodites but would more correctly be termed intersexes. Whilst there is a complete spectrum of phenotypic sexual development within a population of such animals, the most common form is that of a putative female with a prominent up-turned vulva. Reflection of the vulval lips reveals a much-enlarged clitoris. There may be scrotal development, in conjunction with an enlarged penile and preputial sheath. Coarse hair and incipient tusk development may further indicate differing degrees of masculinization. Surgical exploration of intersex animals confirms a complete spectrum of gonadal types, ranging from 2 ovaries with a proportion of testicular tissue in one of them (i.e., an ovotestis) to 2, much-enlarged testicular-like structures with no detectable ovarian tissue. The gonads usually remain within the abdomen, but those with testicular tissue may descend to an inguinal or even scrotal location. The genital tract invariably comprises a bicornuate uterus, a partially vestigial Fallopian tube, and some development of one or both Wolffian ducts adjoining an ovotestis or testicular-like structure to form a convoluted epididymis. Spermatozoa are never present, either in abdominal or scrotal testicular tissue, nor are there any germ cells within the seminiferous tubules, only Sertoli-like cells. Due to the spectrum of gonadal types, sexual behaviour ranges from male-type aggressivity on the one hand to regular oestrous cycles on the other, with periods of standing oestrus during which intromission may be achieved. In animals with functional ovarian tissue in both gonads, foetal development has been observed, at least until days 25-30 of gestation. Almost all intersex pigs possess XX sex chromosomes and usually 36 autosomes; only a very small proportion are chimaeras or mosaics. Chromosome banding techniques have failed to demonstrate a portion of the Y chromosome translocated onto an X chromosome nor has molecular probing revealed the presence of the sex determining gene Sry or other classical Y-related DNA sequences, except in one instance. Breeding records suggest that the intersex condition results most frequently from the influence of an autosomal recessive gene carried by certain boars. Identification of such boars is therefore essential, as the incidence of intersexuality in their offspring may reach 4-5% or more. In terms of the pig industry, economic losses may result from: 1. Lack of fertility in intersex animals. 2. Aggressive behaviour in groups of growing/fattening pigs. 3. Boar taint in the carcase of animals possessing ovotestis. 4. Propagation of the deleterious condition, either by mating or more widely by artificial insemination.

Animals↗